Pyrazoline bearing benzimidazoles: Search for anticancer agent

Article abstract of DOI:10.1016/j.ejmech.2009.09.032

2-acetyl benzimidazole was allowed to react with substituted aromatic aldehydes to get desired intermediate chalcones (2a-g), the cyclocondensation of these intermediates with hydrazine hydrate and phenyl hydrazine in two separate reactions yielded pyrazoline derivatives (3a-g & 4a-g respectively). Among the synthesize compounds, six compounds were granted NSC code and screened at National Cancer Institute (NCI), USA for anticancer activity at a single high dose (10^-5 M) in full NCI 60 cell panel. Among the selected compounds, (4f) 2-[5-(3,4-dimethoxyphenyl)-1-phenyl-4,5-dihydro-1H-3-pyrazolyl]-1H-benzimidazole (NSC 748326) was found to be the most active candidate of the series and selected for further evaluation at five dose level screening.

Full text of DOI:10.1016/j.ejmech.2009.09.032

European Journal of Medicinal Chemistry 45 (2010) 114–119
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Pyrazoline bearing benzimidazoles: Search for anticancer agent
*
Mohammad Shaharyar , M.M. Abdullah, M.A. Bakht, Jaseela Majeed
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Hamdard University, New Delhi-110062, India
a r t i c l e i n f o
a b s t r a c t
Article history:
2-acetyl benzimidazole was allowed to react with substituted aromatic aldehydes to get desired inter-
mediate chalcones (2a–g), the cyclocondensation of these intermediates with hydrazine hydrate and
phenyl hydrazine in two separate reactions yielded pyrazoline derivatives (3a–g & 4a–g respectively).
Among the synthesize compounds, six compounds were granted NSC code and screened at National
Cancer Institute (NCI), USA for anticancer activity at a single high dose (10ꢀ5 M) in full NCI 60 cell panel.
Among the selected compounds, (4f) 2-[5-(3,4-dimethoxyphenyl)-1-phenyl-4,5-dihydro-1H-3-pyr-
azolyl]-1H-benzimidazole (NSC 748326) was found to be the most active candidate of the series and
selected for further evaluation at five dose level screening.
Received 20 February 2009
Received in revised form
8 September 2009
Accepted 17 September 2009
Available online 23 September 2009
Keywords:
Pyrazoline
Benzimidazole
Anticancer activity
Antiproliferative activity
NCI
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
Cancer Institute (NCI), USA for antiproliferative activity at a single
high dose (10ꢀ5 M) in full NCI 60 cell panel. Compound (4f) NSC
Cancer is becoming the biggest health hazard for the world.
Development of resistance against the existing anticancer drugs
keeps research window open in search of newer anticancer mole-
cules. But the window passage has become narrower because it is
rather more difficult to search a molecule that can selectively
inhibit the proliferation of abnormal cells only with least or no
affect on normal cells. Therefore, the search for anticancer agents
continues at various laboratories. Recently, different authors
worldwide have reported antitumor, antiproliferative or anticancer
potential of benzimidazole [1–4] and pyrazoline derivatives [5,6]. It
gave us immense confidence to carry out work on pyrazoline with
antitubercular and antiproliferative activity [7–11]. Due to great
potential of both the moieties, synthesis of pyrazoline bearing
benzimidazole [12a–f] was carried out to evaluate their antituber-
cular [13] & anticancer potential. The data of these synthesized
compounds (except 2g, 3g, 4g) was submitted to National Cancer
Institute (NCI) for antiproliferative activity. As per the protocol of
NCI, only six representative compounds of the series were selected
and granted NSC codes Viz; NSC 747363, NSC 747364, NSC 747365,
NSC 747366, NSC 747367, NSC 748326 and screened at National
748326 exhibited the best result at single dose and was selected for
further evaluation at five dose level screening. This paper presents
in vitro anticancer profile of all the selected compounds including
the most potent compound of the series (4f), 2-[5-(3,4-dimethoxy-
phenyl)-1-phenyl-4,5-dihydro-1H-3-pyrazolyl]-1H-benzimidazole
(NSC 748326), against NCI’s 60 human cancer cell lines.
2. Chemistry
The syntheses of 2-{5-[(substituted) phenyl]-4,5-dihydro-1H-3-
pyrazolyl}-1H-benzoimidazole and 2-{5-[(substituted) phenyl]-1-
phenyl-4,5-dihydro-1H-3-pyrazolyl}-1H-benzimidazole (3a–g)
&
(4a–g) respectively, described in this study are outlined in Scheme 1
and physical data is presented in Table 1. The chalcones (2a–g) were
prepared by reacting 2-acetyl benzimidazole with appropriate
aldehydes in the presence of a base by conventional Claisen-Schmidt
condensation. Reaction between newly synthesized chalcones with
hydrazine hydrate and phenyl hydrazine in ethanol separately gave
pyrazoline and phenyl pyrazoline derivatives in good yield. The
compounds were recrystallized from ethanol. The purity of
the compounds was checked by TLC. Spectral data 1H NMR, IR of all
the synthesized compounds and Mass spectra of selected
compounds were recorded and found in full agreement with the
proposed structures. The elemental analysis results were within
ꢁ0.4% of the theoretical values.
* Corresponding author.
E-mail address: yarmsy@rediffmail.com (M. Shaharyar).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.09.032

M. Shaharyar et al. / European Journal of Medicinal Chemistry 45 (2010) 114–119
115
NH2
NH2
was discarded, and the plates were washed five times with tap
water and air-dried. Sulforhodamine B (SRB) solution (100 L) at
HOOC
m
OH
+
0.4% (w/v) in 1% acetic acid was added to each well, and plates were
incubated for 10 min at room temperature. After staining, unbound
dye is removed by washing five times with 1% acetic acid and the
plates were air-dried. Bound stain was subsequently solubilized
with 10 mM trizma base, and the absorbance was read on an
automated plate reader at a wavelength of 515 nm. For suspension
cells, the methodology was the same except that the assay was
terminated by fixing settled cells at the bottom of the wells by
H3C
Reflux
OH
N
N
H
CH3
CH3COOH
gently adding 50 mL of 80% TCA (final concentration, 16% TCA).
Chromium trioxide
Using the seven-absorbance measurements [time zero, (Tz), control
growth, (C), and test growth in the presence of drug at the five
concentration levels (Ti)], the percentage growth was calculated at
each of the drug concentration levels. Percentage growth inhibition
calculated as:
O
N
N
H
CH3
(1)
RCHO
Ethanol, NaOH
½ðTi—TzÞ=ðC—TzÞꢃ 3 100 for concentrations for which Ti‡ Tz
O
N
½ðTi—TzÞ=Tzꢃ 3 100 for concentrations for which Ti< Tz
H
α
N
H
(2a-g)
Three dose response parameters (GI50, TGI and LC50) were
calculated for each experimental agent. Growth inhibition of 50%
(GI50) was calculated from [(Ti–Tz)/(C–Tz)] ꢄ 100 ¼ 50, which was
the drug concentration resulting in a 50% reduction in the net
protein increase (as measured by SRB staining) in control cells
during the drug incubation. The drug concentration resulting in
total growth inhibition (TGI) was calculated from Ti ¼ Tz. The LC50
(concentration of drug resulting in a 50% reduction in the measured
protein at the end of the drug treatment as compared to that at the
beginning) indicating a net loss of cells following treatment was
calculated from [(Ti–Tz)/Tz] ꢄ 100 ¼ ꢀ50. Values were calculated
for each of these three parameters if the level of activity was
reached; however, if the effect was not reached or was exceeded,
the value for that parameter was expressed as greater or less than
the maximum or minimum concentration tested [14–16].
H
β
R
C6H5.NHNH2
Ethanol
Ethanol
NH2NH2.2H2O
N
N
N
N
N
NH
Hx
Hx
R
N
H
N
H
R
Ha
Hb
Ha
Hb
(3a-g)
(4a-g)
R= Phenyl, 4-Methoxyphenyl; 4-Chlorophenyl; 4-Bromophenyl; 4-Fluorophenyl; 3,4-
Dimethoxy phenyl; 2,6-Dichloro phenyl
Scheme 1. Protocol of synthesis.
3. Pharmacology
4. Results and discussion
3.1. In vitro cancer screen
4.1. Chemistry
The human tumor cell lines of the cancer-screening panel were
grown in RPMI 1640 medium containing 5% fetal bovine serum and
The compounds (2a–g) in 1H NMR spectrum exhibited two
doublets with J values between 15.6–16.8 Hz conforming the trans
2 mM
L
-glutamine. For a typical screening experiment, cells were
L at plating
inoculated into 96 well microtiter plates in 100
m
coupling. The desired pyrazoline derivatives (3a–g
& 4a–g)
densities ranging from 5,000 to 40,000 cells/well depending on the
doubling time of individual cell lines. After cell inoculation, the
microtiter plates were incubated at 37 ꢂC, 5% CO2, 95% air and 100%
relative humidity for 24 h prior to addition of experimental drugs.
After 24 h, two plates of each cell line were fixed in situ with TCA, to
represent a measurement of the cell population for each cell line at
the time of drug addition (Tz). Experimental drugs were solubilized
in dimethyl sulfoxide at 400-fold the desired final maximum test
concentration and stored frozen prior to use. At the time of drug
addition, an aliquot of frozen concentrate was thawed and diluted
to twice the desired final maximum test concentration with
complete medium containing 50
four,10-fold or ½ log serial dilutions were made to provide a total of
five drug concentrations plus control. Aliquots of 100 L of these
different drug dilutions were added to the appropriate microtiter
wells already containing 100 L of medium, resulting in the
obtained from their respective chalcones (2a–g) were characterized
by appearance of three distinct and characteristic double doublets
each with an integration equal to one proton in the aliphatic region.
Among these three double doublets, two exhibited nearly identical
J values and were assigned one for each of the 2 protons of pyr-
azoline at C-4 (methylene) whereas the third double doublet with
different J value assigned to one proton at C-5 of pyrazoline.
4.2. Pharmacological (in vitro anticancer activity)
m
g/mL gentamicin. Additional
4.2.1. Primary single high dose (10ꢀ5 M) full NCI 60 cell
panel in vitro assay
m
All theselected compounds submitted to NationalCancer Institute
(NCI) for in vitro anticancer assay were evaluated for their anticancer
activity. Primary in vitro one dose anticancer assay was performed in
full NCI 60 cell panel representing leukemia, melanoma and cancers
of lung, colon, brain, breast, ovary, kidney and prostate in accordance
with the protocol of the NCI, USA. The compounds were added at
a single concentration (10ꢀ5 M) and the culture was incubated for
48 h. End point determinations were made with a protein binding
dye, sulforhodamine B. Results for each compound were reported as
m
required final drug concentrations. Following drug addition, the
plates were incubated for an additional 48 h at 37 ꢂC, 5% CO2, 95%
air, and 100% relative humidity. For adherent cells, the assay was
terminated by the addition of cold TCA. Cells were fixed in situ by
the gentle addition of 50
tration, 10% TCA) and incubated for 60 min at 4 ꢂC. The supernatant
mL of cold 50% (w/v) TCA (final concen-

116
M. Shaharyar et al. / European Journal of Medicinal Chemistry 45 (2010) 114–119
Table 1
Physical constants of the synthesized compounds.
Compound code
NSC Number
R
Molecular Formula
16H12N2O
Molecular Weight
Melting Point ꢂC
2a
2b
2c
2d
2e
2f
2g
3a
3b
3c
3d
3e
3f
3g
4a
4b
4c
4d
4e
4f
–
–
–
–
–
–
–
–
Phenyl
C
248
278
282
327
266
308
317
262
292
296
341
280
322
331
338
368
372
417
356
398
407
194–196
184–188
202–204
224–226
182–184
196–198
124–126
140–142
234–236
168–170
160–162
152–154
136–138
>250
136–137
120–124
138–140
142–144
128–130
130–134
>250
4-methoxyphenyl
4-chlorophenyl
4-bromophenyl
4-fluorophenyl
3,4-dimethoxy phenyl
2,6-dichlorophenyl
Phenyl
4-methoxyphenyl
4-chlorophenyl
4-bromophenyl
4-fluorophenyl
3,4-dimethoxy phenyl
2,6-dichlorophenyl
Phenyl
4-methoxyphenyl
4-chlorophenyl
4-bromophenyl
4-fluorophenyl
3,4-dimethoxy phenyl
2,6-dichlorophenyl
C17H14N2O2
C16H11N2OCl
C16H11N2OBr
C16H11N2OF
C18H16N2O3
C16H10Cl2N2O
C16H14N4
747363
747364
–
–
–
–
747365
747366
–
C17H16N4O
C16H13N4Cl
C16H13N4Br
C16H13N4F
C18H18N4O2
C16H12Cl2N4
C22H18N4
C23H20N4O
C22H17N4Cl
C22H17N4Br
C22H17N4F
C24H22N4O2
C22H16Cl2N4
–
747367
748326
–
4g
(–) Compounds not selected for antiproliferative activity.
a mean graph of the percent growth of the treated cells when
compared to the untreated control cells. There after obtaining the
results for one dose assay, analysis of historical Developmental
Therapeutics Program (DTP) was performed & compound (NSC-
748326) which satisfied pre-determined threshold inhibition criteria
was selected for NCI full panel 5 dose assay.
and SR were found to be insensitive at the highest tested concen-
tration i.e. 100
concentration.
mM therefore a sign of ‘‘>’’ is used as prefix to the
With regard to the sensitivity against some individual cell lines
(Table 2) the compound showed high activity against Leukemia
CCRF-CEM and RPMI-8226 cell lines with GI50 2.23 & 2.76
respectively. Obtained data revealed an obvious sensitivity profile
towards Colon subpanel (GI50 value ranging from 3.94–12.4 M),
mM
4.2.2. In vitro 5 dose full NCI 60 cell panel assay
m
All the cell lines (about 60), representing nine tumor subpanels,
were incubated at five different concentrations (0.01, 0.1, 1, 10 &
least for HCT-116 and maximum for HCC2998 cell line. The
compound proved to be sensitive towards all the tested Melanoma
100
m
M). The outcomes were used to create log concentration Vs %
Cancer cell lines with not more than 16.1
for the UACC-257 cell line (GI50 77.1
inhibitory activity was observed against the Ovarian IGROVI cancer
cell line with GI50 less than 1 M.
m
M concentrations except
growth inhibition curves and three response parameters (GI50, TGI
and LC50) were calculated for each cell line. The GI50 value (growth
inhibitory activity) corresponds to the concentration of the
compound causing 50% decrease in net cell growth, the TGI value
(cytostatic activity) is the concentration of the compound resulting
in total growth inhibition and LC50 value (cytotoxic activity) is the
concentration of the compound causing net 50% loss of initial cells
at the end of the incubation period of 48 h.
Compound under investigation (NSC-748326) exhibited remark-
able anticancer activity against most of the tested cell lines repre-
senting nine different subpanels with GI50 values between
‘‘0.92–33.1
subpanel showing GI50 at a concentration of 77.1
as three cell lines of Leukemia subpanel namely HL-60 (TB), K-562
mM). The highest growth
m
The criterion for selectivity of a compound depends upon the
ratio obtained by dividing the full panel MID (the average sensi-
tivity of all cell lines towards the test agent) by their individual
subpanel MID (the average sensitivity of all cell lines of a particular
subpanel towards the test agent). Ratios between 3–6 refer to
moderate selectivity, ratios greater than 6 indicate high selectivity
towards the corresponding cell line, while compounds not meeting
either of these criteria rated non-selective [6]. As per this criterion,
compound in the study was found to be mild selective towards
breast cancer subpanel only. Whereas on overall basis it was found
to be non-selective with GI50 ratios ranging from 0.3 to 2.9.
mM’’, except one UACC-257 Cell line of Melanoma
mM (Table 2), where

M. Shaharyar et al. / European Journal of Medicinal Chemistry 45 (2010) 114–119
117
Table 2
the basis of results obtained it was found that 2-[5-(3,4-dime-
thoxyphenyl)-1-phenyl-4,5-dihydro-1H-3-pyrazolyl]-1H-benz-
imidazole (4f; NSC 748326) was most active compound of the
series. Based on close examination on substitutions, it may be
concluded that the role of electron donating groups (–OCH3) on the
phenyl ring at 5th position of pyrazoline has great influence on
anticancer activity. We had also noticed similar role of electron
donating group on antiproliferative activity, although the
compound did not exhibit very good activity but remarkable
superiority within the series when electron doting substituent was
present (unpublished data of our lab). This assumption is further
supported by the presence of electron donating groups on the
phenyl ring particularly (–OCH3) in the structure of Com-
bretastatin-A4 and its pyrazoline derivatives [5]. Finally it is
conceivable that further derivatization of such compounds will be
of interest with the hope to get more selective anticancer agents.
NCI DTP in vitro testing results of compound (NSC 748326) in mM.
Panel
Cell Line
GI50
TGI
LC50
88.7
Concentration Subpanel
per cell line
MIDb
Leukemia
CCRF-CEM
HL-60(BT)
K-562
MOLT-4
RPMI-8226
SR
2.23
>100
>100
17.9
2.76
>100
17.1
9.83
19.9
3.89
15.3
16
21.3
13.6
5.46
9.88
12.4
3.94
4.1
9.91
5.14
6.45
21.3
5.98
15
26.6
11
7.67
5.1
7.47
16.1
4.15
13.1
2.62
77.1
2.59
0.92
4.17
9.31
26.7
19.9
19
53.82
6.82
>100 >100
>100 >100
>100 >100
10.3
76.6
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
Non-Small Cell
Lung Cancer
A549/ATCC
EKVX
13.6
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
COLON 205
HCC2998
HCT-116
HCT-15
HT29
KM12
SW-620
SF-268
SF-295
40
>100
>100
>100
55
49.8
70.9
61.4
52.2
65.7
34.8
33.2
26.1
16.5
20.5
24.7
19.5
23.3
6. Experimental
Colon Cancer
7.4
6.1. Chemistry
All the chemicals used were laboratory grade and procured from
E. Merck (Germany) and S.D. Fine Chemicals (India). Melting points
were determined by open tube capillary method and are uncor-
rected. Thin layer chromatography (TLC) plates, (silica gel G) were
used to confirm the purity of commercial reagents used,
compounds synthesized and to monitor the reactions as well. Two
different solvent systems; toluene: ethyl acetate: formic acid
(5:4:1) and petroleum ether: toluene: acetic acid (5:4:1), were used
to run the TLC. The spots were located under iodine vapors/UV
light. IR spectra were obtained on a Perkin-Elmer 1720 FT-IR
spectrometer (KBr Pellets). 1H NMR spectra were recorded on
a Bruker AC 400 MHz, spectrometer-using TMS as internal standard
in DMSO-d6/CDCl3. The FAB mass spectra were recorded on a JEOL
SX 102/DA-6000 Mass Spectrometer.
CNS Cancer
14.6
>100 >100
51.4
38.6
>100
99.5
SF-539
SNB-19
SNB-75
U251
>100 >100
47.4
80.2
21.7
92.7
>100 >100
>100 >100
39
15.1
>100 >100
>100 >100
19.1
18.9
>100 >100
>100 >100
>100 >100
94.5
>100 >100
>100 >100
>100
>100
62.9
Melanoma
Cancer
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
IGROVI
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
786-0
16.04
>100
>100
67.6
Ovarian
Cancer
16.16
11.94
>100
62.2
6.1.1. General procedure for synthesis of 1-(1H-benzimidazol-2-yl)-
3-[(substituted) phenyl]-2-propen-1-one (2a–g)
According to Claisen–Schmidt condensation, 2-acetyl benz-
imidazole was allowed to react with substituted aromatic alde-
hydes in 10% ethanolic NaOH solution to obtain the desired
chalcones derivatives.
>100
33.1
24.2
5.14
14
Renal Cancer
A498
ACHN
58.1
>100 >100
>100
CAKI-1
RXF 393
SN12C
TK-10
UO-31
3.6
24.7
36.1
>100 >100
65.9
39.8
49
>100 >100
15.3
91.5
>100
97
6.1.1.1. 1-(1H-benzimidazol-2-yl)-3-phenyl-2-propen-1-one (2a). IR:
(KBr, cmꢀ1)3251(N–H),1661(C]O),1594(C]N),1329(C–N).1HNMR
(DMSO-d6): 6.68 (1H, t, J ¼ 7.6 Hz, Ar–H), 6.92 (1H, d, J ¼ 16.8 Hz, Ha),
7.11 (1H, d, J ¼ 16.4 Hz, Hb), 7.20–7.32 (6H, m, Ar–H), 7.42 (1H, d,
J ¼ 8.0 Hz, Ar–H), 7.50 (1H, d, J ¼ 8.0 Hz, Ar–H), 11.92 (1H, s, NH
benzimidazole). Calcd/Anal. [C77.40/77.12, H4.87/4.88, N11.28/11.25].
13.4
17.3
15.7
2.2
6.27
33.1
2.41
>100
>100
>100
Prostate Cancer PC-3
DU-145
MCF7
19.69
5.74
Breast Cancer
54.2
>100
MDA-MB-231/ 7.19
ATCC
HS 578T
BT-549
T-47D
MDA-MB-468
6.1.1.2. 1-(1H-benzimidazol-2-yl)-3-(4-methoxyphenyl)-2-propen-1-
one (2b). IR: (KBr, cmꢀ1) 3261 (N–H), 1653 (C]O), 1576 (C]N),
1330 (C–N). 1H NMR (CDCl3): 3.63 (3H, s, OCH3), 7.01(2H, d,
J ¼ 7.6 Hz, Ar–H), 7.32 (4H, m, Ar–H), 7.46 (1H, d, J ¼ 16.4 Hz, Ha),
7.54 (1H, d, J ¼ 16.0 Hz, Hb), 7.81 (1H, d, J ¼ 8.0 Hz, Ar–H), 7.85 (1H,
d, J ¼ 7.6 Hz, Ar–H), 13.11 (1H, s, NH benzimidazole). Calcd/Anal. [C
73.37/73.52, H 5.07/5.06, N 10.07/10.08].
6.18
10.3
3.09
5.25
16.87
41.2
69
51.1
21.2
>100
>100
>100
64.9
MIDa
a
MID ¼ Average sensitivity of all cell line of a particular subpanel in
mM.
b
MID ¼ Average sensitivity of all cell line (about 60) in
mM.
6.1.1.3. 1-(1H-benzimidazol-2-yl)-3-(4-chlorophenyl)-2-propen-1-one
(2c). IR: (KBr, cmꢀ1) 3266 (N–H), 1653 (C]O), 1578 (C]N), 1332
(C–N), 771 (C–Cl). 1H NMR (DMSO-d6): 7.28 (2H, d, J ¼ 7.6 Hz, Ar–H),
7.48 (4H, m, Ar–H), 7.62 (1H, d, J ¼ 16.0 Hz, Ha), 7.68 (1H, d,
J ¼ 16.0 Hz, Hb), 7.72 (1H, d, J ¼ 8.0 Hz, Ar–H), 7.75 (1H, d, J ¼ 7.6 Hz,
Ar–H), 11.80 (1H, s, NH benzimidazole). FAB MASS m/e: 283(Mþ1).
Calcd/Anal. [C 67.97/68.12, H 3.92/3.91, N 9.91/9.9].
5. Conclusion
Cyclocondensation of hydrazine hydrate & phenyl hydrazine
with 2-acetyl benzimidazole derived chalcones (2a–g), yielded
respective pyrazoline derivatives 3a–g & 4a–g. All the compounds
were submitted to screen for their anticancer potential at NCI on

118
M. Shaharyar et al. / European Journal of Medicinal Chemistry 45 (2010) 114–119
6.1.1.4. 1-(1H-benzimidazol-2-yl)-3-(4-bromophenyl)-2-propen-1-one
(2d). IR: (KBr, cmꢀ1) 3261 (N–H), 1653 (C]O), 1576 (C]N), 1378
(C–N). 1H NMR (DMSO-d6): 7.03–7.12 (4H, m, Ar–H), 7.32 (2H,
d, J ¼ 8.0 Hz, Ar–H), 7.36 (1H, d, J ¼ 8.0 Hz, Ar–H), 7.39 (1H, d,
J ¼ 8.0 Hz, Ar–H), 7.51 (1H, d, J ¼ 16.0 Hz, Ha), 7.55 (1H, d,
J ¼ 16.4 Hz, Hb), 13.11 (1H, s, NH benzimidazole). Calcd/Anal. [C
58.74/58.75, H 3.39/3.39, N 8.56/8.56].
N). 1H NMR (DMSO-d6): 3.01 (1H, dd, J ¼ 8.4, 8.0 Hz, Ha), 3.76 (1H,
dd, J ¼ 13.2, 13.2 Hz, Hx), 4.41 (1H, dd, J ¼ 8.0, 8.0 Hz, Hb), 6.73 (2H,
d, J ¼ 7.6 Hz, Ar–H), 7.28 (5H, m, Ar–H), 7.38 (1H, d, J ¼ 8.0 Hz, Ar–
H), 11.21 (1H, s, NH benzimidazole), (1H, NH pyrazoline missing).
Calcd/Anal. [C 56.32/56.51, H 3.84/3.99, N 16.42/16.46].
6.1.2.5. 2-[5-(4-fluorophenyl)-4,5-dihydro-1H-3-pyrazolyl]-1H-benz-
imidazole (3e). IR: (KBr, cmꢀ1) 3008 (N–H), 1622 (C]N), 1330 (C-
N). 1H NMR (DMSO-d6): 3.12 (1H, dd, J ¼ 8.0, 8.0 Hz, Ha), 3.92 (1H,
dd, J ¼ 13.6, 13.6 Hz, Hx), 4.23 (1H, dd, J ¼ 7.6, 8.0 Hz, Hb), 6.67 (2H,
d, J ¼ 8.0 Hz, Ar–H), 6.85 (2H, d, J ¼ 8.0 Hz, Ar–H), 6.91 (3H, m, Ar–
H), 7.32 (1H, d, J ¼ 7.6, Hz, Ar–H) 12.02 (1H, s, NH benzimidazole),
(1H, NH pyrazoline missing). Calcd/Anal. [C 68.56/68.67, H 4.67/
4.66, N 19.99/19.98].
6.1.1.5. 1-(1H-benzimidazol-2-yl)-3-(4-fluorophenyl)-2-propen-1-one
(2e). IR: (KBr, cmꢀ1) 3260 (N–H), 1650 (C]O), 1577 (C]N), 1335
(C–N). 1H NMR (DMSO-d6): 6.89-7.20 (7H, m, Ar–H), 7.28 (1H, d,
J ¼ 15.6 Hz, Ha), 7.32 (1H, d, J ¼ 15.6 Hz, Hb), 7.38 (1H, d, J ¼ 7.6 Hz,
Ar–H), 7.41 (1H, d, J ¼ 8.0 Hz, Ar–H), 12.80 (1H, s, NH benzimid-
azole). Calcd/Anal. [C 72.17/71.98, H 4.16/4.15, N 10.52/10.51].
6.1.1.6. 1-(1H-benzimidazol-2-yl)-3-(3,4-dimethoxyphenyl)-2-propen-
1-one (2f). IR: (KBr, cmꢀ1) 3261 (N–H), 1653 (C]O), 1508 (C]N),
1330 (C–N). 1H NMR (DMSO-d6): 3.59 (6H, s, 2 ꢄ OCH3), 6.68–6.72
(3H, m, Ar–H), 6.74 (1H, d, J ¼ 8.0 Hz, Ar–H), 6.79 (1H, d, J ¼ 7.6 Hz,
Ar–H), 7.26 (1H, d, J ¼ 16.0 Hz, Ha), 7.32 (1H, d, J ¼ 8.0 Hz, Ar–H), 7.36
(1H, d, J ¼ 16.0 Hz, Hb), 7.42 (1H, d, J ¼ 8.0 Hz, Ar–H), 11.91 (1H, s, NH
benzimidazole). Calcd/Anal. [C 70.12/70.05, H 5.23/5.24, N 9.09/9.09].
6.1.2.6. 2-[5-(3,4-dimethoxyphenyl)-4,5-dihydro-1H-3-pyrazolyl]-
1H-benzimidazole (3f). IR: (KBr, cmꢀ1) 3003(N–H), 1609(C]N),
1332(C–N). 1H NMR (DMSO-d6): 3.43 (1H, dd, J ¼ 8.4, 8.0 Hz, Ha),
3.68 (6H, s, 2 ꢄ OCH3), 4.21 (1H, dd, J ¼ 13.2, 13.6 Hz, Hx), 6.13 (1H,
dd, J ¼ 8.0, 8.0 Hz, Hb), 6.64 (5H, m, Ar–H), 6.70 (1H, d, J ¼ 7.6 Hz,
Ar–H), 6.75 (1H, d, J ¼ 7.6 Hz, Ar–H), 11.12 (1H, s, NH benzimid-
azole), (1H, NH pyrazoline missing). Calcd/Anal. [C 67.07/67.00, H
5.63/5.63, N 17.38/17.40].
6.1.1.7. 1-(1H-benzimidazol-2-yl)-3-(2,6-dichlorophenyl)-2-propen-
1-one (2g). IR: (KBr, cmꢀ1) 3258 (N–H), 1653 (C]O), 1508 (C]N),
766 (C–Cl), 1332 (C–N). 1H NMR (DMSO-d6): 7.35 (2H, s, Ar–H), 7.42
(1H, t, J ¼ 8.4, 7.6 Hz, Ar–H), 7.59-7.71 (4H, m, Ar–H), 7.94 (1H, d,
J ¼ 16.4 Hz, Ha), 8.16 (1H, d, J ¼ 16.4 Hz, Hb), 13.59 (1H, s, NH benz-
imidazole). Calcd/Anal. [C 60.59/60.57, H 3.18/3.17, N 8.83/8.83].
6.1.2.7. 2-[5-(2,6-dichlorophenyl)-4,5-dihydro-1H-3-pyrazolyl]-1H-
benzimidazole (3g). IR: (KBr, cmꢀ1) 3003 (N–H), 1608 (C]N), 1332
(C–N), 766 (C–Cl). 1H NMR (CDCl3): 3.48 (1H, dd, J ¼ 8.8, 8.4 Hz, Ha),
3.88 (1H, dd, J ¼ 13.2, 13.6 Hz, Hx), 6.26 (1H, dd, J ¼ 8.8, 8.8 Hz, Hb),
7.12 (1H, t, J ¼ 8.0, 8.0 Hz, Ar–H), 7.23–7.36 (4H, m, Ar–H), 7.48 (1H,
d, J ¼ 8.0 Hz, Ar–H), 7.79 (1H, d, J ¼ 7.6 Hz, Ar–H), 10.01 (1H, s, NH
benzimidazole), (1H, NH pyrazoline missing). FAB MASS m/e:
331(Mþ). Calcd/Anal. [C 58.02/58.00, H 3.65/3.65, N 16.92/16.91].
6.1.2. General procedure for synthesis of 2-{5-[(substituted)
phenyl]-4,5-dihydro-1H-3-pyrazolyl}-1H-benzoimidazole (3a–g)
To an ethanolic solution of chalcones (2a–g), hydrazine hydrate
was added drop wise. The reaction mixture was heated under reflux
for 6 h and then cooled and poured onto crushed ice. The solid
pyrazoline product was filtered and re-crystallized from ethanol.
6.1.3. General procedure for synthesis of 2-{5-[(substituted)
phenyl]-1-phenyl-4,5-dihydro-1H-3-pyrazolyl}-1H-benzimidazole
(4a–g)
To a solution of chalcones (2a–g) in ethyl alcohol, phenyl
hydrazine was added drop wise. The reaction mixture was heated
under reflux for 12 h and then cooled and poured onto crushed ice.
The solid pyrazoline product was filtered and re-crystallized.
6.1.2.1. 2-(5-phenyl-4,5-dihydro-1H-3-pyrazolyl)-1H-benzimidazole
(3a). IR: (KBr, cmꢀ1) 3103 (N–H), 1638 (C]N), 1330 (C–N). 1H NMR
(DMSO-d6): 3.21 (1H, dd, J ¼ 8.4, 8.4 Hz, Ha), 4.02 (1H, dd, J ¼ 13.2,
13.2 Hz, Hx), 5.81 (1H, dd, J ¼ 8.0, 8.0 Hz, Hb), 6.82 (4H, m, Ar–H),
6.98 (1H, d, J ¼ 8.0 Hz, Ar–H), 7.31 (1H, d, J ¼ 8.0 Hz, Ar–H), 7.35 (3H,
m, Ar–H), 11.01 (1H, s, NH benzimidazole), (1H, NH pyrazoline
missing). Calcd/Anal. [C 73.26/73.51, H 5.38/5.38, N 21.36/21.40].
6.1.3.1. 2-(1,5-diphenyl-4,5-dihydro-1H-3-pyrazolyl)-1H-benzimid-
azole (4a). IR: (KBr, cmꢀ1) 2832 (N–H), 1608(C]N), 1374(C–N). 1H
NMR (DMSO-d6): 3.11 (1H, dd, J ¼ 9.2, 9.2 Hz, Ha), 3.75 (1H, dd,
J ¼ 14.0, 14.0 Hz, Hx), 5.82 (1H, dd, J ¼ 9.2, 9.2 Hz, Hb), 6.62–7.81
(14H, m, Ar–H), 10.52 (1H, s, NH benzimidazole). Calcd/Anal.
[C 78.08/78.01, H 5.36/5.53, N 16.56/16.55].
6.1.2.2. 2-[5-(4-methoxyphenyl)-4,5-dihydro-1H-3-pyrazolyl]-1H-
benzimidazole (3b). IR: (KBr, cmꢀ1) 3047 (N-H), 1608 (C]N),
1332(C–N). 1H NMR (CDCl3): 3.52 (1H, dd, J ¼ 8.8, 8.8 Hz, Ha), 3.58
(3H, s, OCH3), 4.09 (1H, dd, J ¼ 13.6,13.2 Hz, Hx), 6.13 (1H, dd, J ¼ 8.4,
8.4 Hz, Hb), 6.73 (2H, d, J ¼ 8.4 Hz, Ar–H), 7.13 (4H, m, Ar-H), 7.63
(1H, d, J ¼ 8.0 Hz, Ar–H), 7.68 (1H, d, J ¼ 8.0 Hz, Ar–H), 11.87 (1H, s,
NH benzimidazole), (1H, NH pyrazoline missing). FAB MASS m/e:
290(Mꢀ2). Calcd/Anal. [C 69.85/70.08, H 5.52/5.50, N 19.17/19.21].
6.1.3.2. 2-[5-(4-methoxyphenyl)-1-phenyl-4,5-dihydro-1H-3-pyr-
azolyl]-1H-benzimidazole (4b). IR: (KBr, cmꢀ1
) 3047 (N–H),
1582(C]N), 1374(C–N). 1H NMR (DMSO-d6): 3.02 (1H, dd, J ¼ 9.6,
9.6 Hz, Ha), 3.56 (3H, s, OCH3), 3.72 (1H, dd, J ¼ 14.0,14.0 Hz, Hx), 5.91
(1H, dd, J ¼ 9.6, 9.2 Hz, Hb), 6.62 (3H, m, Ar–H), 6.92 (2H, d, J ¼ 8.4 Hz,
Ar–H), 7.21 (1H, d, J ¼ 8.0 Hz, Ar–H), 7.29 (4H, m, Ar–H), 7.33 (1H, d,
J ¼ 8.0 Hz, Ar–H), 7.45 (2H, m, Ar–H),11.21 (1H, s, NH benzimidazole).
Calcd/Anal. [C, 74.98/75.01, H 5.47/5.47, N 15.21/15.22].
6.1.2.3. 2-[5-(4-chlorophenyl)-4,5-dihydro-1H-3-pyrazolyl]-1H-benz-
imidazole (3c). IR: (KBr, cmꢀ1) 3003 (N–H), 1610 (C]N), 1330 (C–
N), 766 (C–Cl). 1H NMR (DMSO-d6): 3.42 (1H, dd, J ¼ 8.4, 8.4 Hz, Ha),
3.63 (1H, dd, J ¼ 13.2, 13.2 Hz, Hx), 4.27 (1H, dd, J ¼ 7.6, 8.0 Hz, Hb),
6.68 (6H, m, Ar–H), 6.81 (1H, d, J ¼ 7.6 Hz, Ar–H), 6.92 (1H, d,
J ¼ 8.0 Hz, Ar–H), 7.32 (1H, d, J ¼ 8.0 Hz, Ar–H), 11.18 (1H, s, NH
benzimidazole), (1H, NH pyrazoline missing). Calcd/Anal. [C 64.76/
65.01, H 4.42/4.43, N 18.88/18.84].
6.1.3.3. 2-[5-(4-chlorophenyl)-1-phenyl-4,5-dihydro-1H-3-pyrazolyl]-
1H-benzimidazole (4c). IR: (KBr, cmꢀ1
)
3003 (N–H), 1582
(C]N),1332 (C–N), 738 (C–Cl). 1H NMR (DMSO-d6): 3.41 (1H, dd,
J ¼ 9.6, 9.2 Hz, Ha), 3.58 (1H, dd, J ¼ 13.6, 13.6 Hz, Hx), 5.81 (1H, dd,
J ¼ 9.2, 9.2 Hz, Hb), 6.66 (5H, m, Ar–H), 6.72 (1H, d, J ¼ 8.4 Hz, Ar–H),
6.78 (3H, m, Ar–H), 6.82 (1H, d, J ¼ 8.4 Hz, Ar–H), 6.91-7.21 (3H, m,
Ar–H), 9.82 (1H, s, NH benzimidazole). Calcd/Anal. [C 70.87/70.88,
H 4.60/4.61, N 15.03/15.03].
6.1.2.4. 2-[5-(4-bromophenyl)-4,5-dihydro-1H-3-pyrazolyl]-1H-benz-
imidazole (3d). IR: (KBr, cmꢀ1) 3112 (N–H), 1622 (C]N), 1333 (C–

M. Shaharyar et al. / European Journal of Medicinal Chemistry 45 (2010) 114–119
119
6.1.3.4. 2-[5-(4-bromophenyl)-1-phenyl-4,5-dihydro-1H-3-pyrazolyl]-
1H-benzimidazole (4d). IR: (KBr, cmꢀ1) 3014 (N–H), 1608 (C]N),
1328 (C–N). 1H NMR (DMSO-d6): 3.26 (1H, dd, J ¼ 9.2, 9.2 Hz, Ha),
3.82 (1H, dd, J ¼ 14.0, 13.6 Hz, Hx), 6.11 (1H, dd, J ¼ 9.2, 9.2 Hz, Hb),
6.67 (1H, d, J ¼ 8.0 Hz, Ar–H), 6.78 (2H, d, J ¼ 8.0 Hz, Ar–H), 6.81
(3H, m, Ar–H), 6.88 (1H, d, J ¼ 8.0 Hz, Ar–H), 6.92–7.28 (6H, m, Ar-
H), 11.01 (1H, s, NH benzimidazole). Calcd/Anal. [C 63.32/63.33, H
4.11/4.11, N 13.43/13.44].
screening of the newly synthesized compounds. We are also
grateful to Prof. E. De Clercq, of Rega Institute of Medical Research,
Belgium, for help and necessary suggestions.
Appendix. Supplementary data
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.ejmech.2009.09.032.
6.1.3.5. 2-[5-(4-fluorophenyl)-1-phenyl-4,5-dihydro-1H-3-pyrazolyl]-
1H-benzimidazole (4e). IR:(KBr, cmꢀ1)3004(N–H),1608(C]N),1305
(C–N). 1H NMR (DMSO-d6): 3.30 (1H, dd, J ¼ 9.6, 9.6 Hz, Ha), 3.56 (1H,
dd, J ¼ 14.0, 14.0 Hz, Hx), 4.92 (1H, dd, J ¼ 9.6, 9.6 Hz, Hb), 6.78 (1H, t,
J ¼ 7.6, 7.6 Hz, Ar–H), 6.81(2H, d, J ¼ 7.6 Hz, Ar–H), 6.88(4H, m, Ar–H),
6.91 (2H, d, J ¼ 8.0 Hz, Ar–H), 7.01 (1H, d, J ¼ 7.6 Hz, Ar–H), 7.21(1H, d,
J ¼ 7.6 Hz, Ar–H), 7.28 (2H, m, Ar–H), 9.88 (1H, s, NH benzimidazole).
Calcd/Anal. [C 74.14/74.18, H 4.81/4.80, N 15.72/15.72].
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azolyl]-1H-benzimidazole (4f). IR: (KBr, cmꢀ1
)
3003 (N–H),
1616(C]N), 1374(C–N). 1H NMR (DMSO-d6): 3.36 (1H, dd, J ¼ 9.2,
9.2 Hz, Ha), 3.82 (1H, dd, J ¼ 14.0, 13.6 Hz, Hx), 6.01 (1H, dd, J ¼ 9.2,
9.6 Hz, Hb), 6.76 (1H, d, J ¼ 7.6 Hz, Ar–H), 6.82 (1H, d, J ¼ 7.6 Hz, Ar–
H), 7.22–7.41 (3H, m, Ar–H), 7.44 (2H, d, J ¼ 8.0 Hz, Ar–H), 7.48–7.57
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azolyl]-1H-benzimidazole (4g). IR: (KBr, cmꢀ1) 3004 (N–H), 1608
(C]N),1374 (C–N), 767 (C–Cl).1H NMR (CDCl3): 3.33 (1H, dd, J ¼ 9.2,
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407(Mþ). Calcd/Anal. [C 64.88/65.01, H 3.96/3.96, N 13.76/13.75].
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The author’s wishes to express there thanks to, staff members of
National Cancer Institute (NCI), USA, for in vitro anti cancer